Toward Improved Therapy for Classic Galactosemia

描述（由申请人提供）：遗传性半乳糖-1-磷酸尿苷酰转移酶缺乏症（GALT，E.C. 2.7.7.12）活动可能导致一种称为经典半乳糖血症（OMIM 230400）的潜在致命疾病。尽管新生儿筛查、早期诊断和半乳糖限制饮食可以挽救生命，但许多经典半乳糖血症患者在以后的生活中会出现并发症，包括生长、神经心理和语言延迟以及卵巢功能不全（POI）。与这种疾病相关的急性致死性和长期并发症的致病机制在很大程度上仍然未知，因此进一步阻碍了更有效治疗的发展。然而，一些证据表明，半乳糖激酶（GALK）的产物半乳糖-1-磷酸（gal-1 P）水平升高是典型半乳糖血症的主要（如果不是唯一）致病机制。因此，我们假设通过抑制GALK来预防gal-1 P的产生将减轻GALT缺陷细胞的半乳糖毒性。为了验证这一假设，我们与NIH化学基因组学中心（NCGC）的高通量筛选（HTS）设施合作，启动了一项定量高通量筛选（qHTS）活动，以鉴定人GALK的小分子抑制剂。到目前为止，我们已经确定了149个小分子化合物，在微摩尔（μ M）浓度，抑制80%或更多的控制GALK活性在体外。通过对这些化合物进行生物化学、结构和基于细胞的表征，我们已经鉴定了几种有希望的化学型，其无毒且有效降低人GALT缺陷型成纤维细胞中的gal-1 P积累。然而，将这些有前途的小分子GALK抑制剂转化为安全和有用的治疗剂需要进一步的化学修饰和在哺乳动物模型中的体内研究。在本申请中，我们提出建立一种创新的、具有成本效益的、迭代的和平行的优化过程，以（1）在体外和基于细胞的测定中改善经验证的GALK抑制剂针对GALK的生物活性和药物样性质;（2）通过评估优化的抑制剂在减少半乳糖激发的GalT敲除小鼠中的gal-1 P产生的功效来进行概念验证（POC）研究。拟议项目的完成将确定一种新靶点GALK的体内化学探针，该靶点具有重要意义，从了解人体器官发育/功能中的半乳糖代谢到开发一种改进的治疗方法，以解决使人衰弱的孤儿病经典半乳糖血症未满足的医疗需求。

DESCRIPTION (provided by applicant): Hereditary deficiency of galactose-1-phosphate uridylyltransferase (GALT, E.C. 2.7.7.12) activity can lead to a potentially lethal disease called Classic Galactosemia (OMIM 230400). Despite the life-saving consequences of newborn screening, early diagnosis, and a galactose-restricted diet, many patients with Classic Galactosemia suffer later in life from complications including growth, neuropsychological, and speech delays as well as premature ovarian insufficiency (POI). The pathogenic mechanisms of the acute lethality and the long-term complications associated with this disorder remain largely unknown, thus further hamper the development of more effective therapies. Yet, several lines of evidence suggested that elevated level of galactose-1-phosphate (gal-1P), product of galactokinase (GALK), is a major, if not sole, pathogenic mechanism in Classic Galactosemia. We, therefore, hypothesize that prevention of gal-1P production by inhibiting GALK will relieve GALT-deficient cells from galactose toxicity. To test this hypothesis, we, in collaboration with th High-throughput Screening (HTS) Facility at the NIH Chemical Genomics Center (NCGC), had launched a quantitative high-throughput screening (qHTS) campaign to identify small molecule inhibitors of the human GALK. To date, we have identified 149 small molecule compounds that, at micromolar (¿M) concentration, inhibit 80% or more of control GALK activity in vitro. Through biochemical, structure- and cell-based characterization of these compounds, we have identified a few promising chemotypes that are non- toxic and effective in lowering gal-1P accumulation in human GALT-deficient fibroblasts. The translation of these promising small molecule GALK inhibitors into safe and useful therapeutics, however, requires further chemical modifications and in vivo studies in mammalian animal models. In this application, we propose to set up an innovative, cost-effective, iterative and parallel optimization process to (1) Improve the biologicl activities and drug-like properties of the validated GALK inhibitors against GALK in vitro and in cell-based assays; (2) perform proof-of-concept (POC) studies by evaluating the efficacy of the optimized inhibitors in reducing gal-1P production galactose-challenged GalT-knockout mice. The completion of the proposed project will identify in vivo chemical probes for a novel target, GALK, which has significant implications ranging from the understanding of galactose metabolism in human organ development/ functions to the development of an improved therapy to address the unmet medical needs of the debilitating orphan disease, Classic Galactosemia.